SUMMARY The filoviruses (family Filoviridae), comprising five ebolaviruses, a cuevavirus, and two marburgviruses, are negative-strand RNA viruses that cause severe hemorrhagic fever with human case fatality rates of up to 90%. The 2014?2016 epidemic in Western Africa has demonstrated the potential for these viruses to cause health emergencies of global scope, and highlighted the need for development of therapeutics and vaccines. Monoclonal antibodies (mAbs) represent an attractive therapeutic modality for filoviruses because mAb cocktails or convalescent IgG has been demonstrated to provide post-exposure protection of non-human primates. Several mAb cocktails that are specific to the Zaire ebolavirus species (EBOV) have advanced to clinical trials. However, mAb therapies against other filoviruses are lacking, and most mAbs are narrowly specific for a single filovirus. Sudan virus (SUDV), Bundibugyo virus (BDBV), and Marburg virus (MARV) have all caused large human outbreaks with high case fatality rates, and thus have similar epidemic potential. Given the sporadic and unpredictable nature of outbreaks, it would be highly advantageous to have a single broad-spectrum anti-filovirus therapeutic. In this proposal, we will utilize innovative protein engineering technologies to develop bispecific antibodies (Bis-mAbs) as cross-protective therapies. In preliminary data, we have developed Bis-mAbs that target conserved ebolavirus epitopes on the virus as well as the universal host endosomal receptor for filoviruses, Niemann-Pick C1 (NPC1). These Bis-mAbs show broad neutralization of ebolavirus glycoprotein-mediated cell entry. In Aim 1, we will test new virus/host Bis-mAb designs to downselect the most promising candidates for mouse challenge experiments. In Aim 2, we will explore an alternate strategy in which novel human mAbs targeting broad ebolavirus epitopes are combined into Bis-mAbs that have the capacity to engage multiple broad viral epitopes simultaneously. In Aim 3, we extend these strategies to include MARV, whose glycoprotein is highly divergent from the ebolaviruses, and explore the potential of entirely host-directed Bis-mAbs with pan-filovirus potential. In all cases, the bifunctional capability of the Bis-mAbs is critical for mechanism of action and thus the broad-spectrum activity cannot be achieved by mixing conventional mAbs together in a cocktail. The most promising broad Bis-mAbs will be tested for their ability to confer protection of mice and guinea pigs from challenge by multiple filoviruses. The unique aspects of antiviral mechanisms will be deciphered by virologic and biochemical experiments. The investigative team includes top experts in antibody engineering, filovirus biology and pathology, and commercialization of antiviral therapeutics. This work will result in new broad-spectrum anti- filovirus therapeutic antibodies that will be ready for advanced preclinical development.